Jo Ann Wise

Preparation and analysis of low molecular weight RNAs and small ribonucleoproteins

Publisher Summary This chapter describes the preparation and analysis of low molecular weight RNAs and small ribonucleoproteins. For routine preparation of total RNA, mechanical breakage can be employed with glass beads in the presence of the chaotropic agent guanidine thiocyanate to obtain high yields of good quality RNA. To determine in which cell compartment the RNA of interest resides, or to enrich for a species if its localization is known, it is often desirable to fractionate cells before extracting RNA. Small cytoplasmic RNAs relatively free of contamination with ribosomal RNAs or their breakdown products can be obtained from a postribosomal supernatant. It is reported that one-dimensional gels provide adequate resolution of both tRNAs and snRNAs in yeast. Therefore a semidenaturing two-dimensional system is adopted for analysis of RNAs labeled either in vivo or in vitro. For bigger RNAs, northern blot analysis is used by electrophoretically transferring the RNA from a high-resolution gel.

A simple high-efficiency method for random mutagenesis of cloned genes using forced nucleotide misincorporation

We describe a simple protocol for introducing random point mutations into cloned DNA fragments via forced misincorporation of deoxynucleoside triphosphates (dNTPs) by either of two polymerases which lack proofreading activity, reverse transcriptase or a mutant T7 DNA polymerase. A high ratio of one nucleotide to the other three is used to enhance the error rate. Mutagenesis is initiated from a specific primer and restricted to a relatively short segment by limiting the amounts of the nonmutagenic dNTPs. Our method is highly efficient, resulting in the isolation of greater than 50% mutant plasmids with most polymerase/forcing dNTP combinations. A wide spectrum of mutations can be obtained, in contrast to commonly used methods for random mutagenesis.

Evolutionary origin of the U6 small nuclear RNA intron.

U6 is the most conserved of the five small nuclear RNAs known to participate in pre-mRNA splicing. In the fission yeast Schizosaccharomyces pombe, the single-copy gene encoding this RNA is itself interrupted by an intron (T. Tani and Y. Ohshima, Nature (London) 337:87-90, 1989). Here we report analysis of the U6 genes from all four Schizosaccharomyces species, revealing that each is interrupted at an identical position by a homologous intron; in other groups, including ascomycete and basidiomycete fungi, as well as more distantly related organisms, the U6 gene is colinear with the RNA. The most parsimonious interpretation of our data is that the ancestral U6 gene did not contain an intron, but rather, it was acquired via a single relatively recent insertional event.

Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p.

Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER). Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP. S. pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E. coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the association of S. pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [35S]-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.